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Live cells, as reservoirs of biochemical reactions, can serve as amazing integrated chemical plants where precursor formation, nucleation and growth of nanocrystals, and functional assembly, can be carried out accurately following an artificial program. It is crucial but challenging to deliberately direct intracellular pathways to synthesize desired nanocrystals that cannot be produced naturally in cells, because the relevant reactions exist in different spatiotemporal dimensions and will never encounter each other spontaneously. This article summarizes the progress in the introduction of inorganic functional nanocrystals into live cells via the 'artificially regulated space-time-coupled live-cell synthesis' strategy. We also describe ingenious bio-applications of nanocrystal-cell systems, and quasi-biosynthesis strategies expanded from live-cell synthesis. Artificially regulated live-cell synthesis-which involves the interdisciplinary application of biology, chemistry, nanoscience and medicine-will enable researchers to better exploit the unanticipated potentialities of live cells and open up new directions in synthetic biology.
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Influenza A virus (IAV) is a global health threat. The cellular endocytic machineries harnessed by IAV remain elusive. Here, by tracking single IAV particles and quantifying the internalized IAV, we found that sphingomyelin (SM)-sequestered cholesterol, but not accessible cholesterol, is essential for the clathrin-mediated endocytosis (CME) of IAV. The clathrin-independent endocytosis of IAV is cholesterol independent, whereas the CME of transferrin depends on SM-sequestered cholesterol and accessible cholesterol. Furthermore, three-color single-virus tracking and electron microscopy showed that the SM-cholesterol complex nanodomain is recruited to the IAV-containing clathrin-coated structure (CCS) and facilitates neck constriction of the IAV-containing CCS. Meanwhile, formin-binding protein 17 (FBP17), a membrane-bending protein that activates actin nucleation, is recruited to the IAV-CCS complex in a manner dependent on the SM-cholesterol complex. We propose that the SM-cholesterol nanodomain at the neck of the CCS recruits FBP17 to induce neck constriction by activating actin assembly. These results unequivocally show the physiological importance of the SM-cholesterol complex in IAV entry. IMPORTANCE IAV infects cells by harnessing cellular endocytic machineries. A better understanding of the cellular machineries used for its entry might lead to the development of antiviral strategies and would also provide important insights into physiological endocytic processes. This work demonstrated that a special pool of cholesterol in the plasma membrane, SM-sequestered cholesterol, recruits FBP17 for the constriction of clathrin-coated pits in IAV entry. Meanwhile, the clathrin-independent cell entry of IAV is cholesterol independent. The internalization of transferrin, the gold-standard cargo endocytosed solely via CME, is much less dependent on the SM-cholesterol complex. These results provide new insights into IAV infection and the pathway/cargo-specific involvement of the cholesterol pool(s).
Assuntos
Colesterol , Vesículas Revestidas por Clatrina , Proteínas de Ligação a Ácido Graxo , Forminas , Vírus da Influenza A , Internalização do Vírus , Actinas/metabolismo , Animais , Colesterol/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Vesículas Revestidas por Clatrina/virologia , Endocitose/fisiologia , Proteínas de Ligação a Ácido Graxo/metabolismo , Forminas/metabolismo , Vírus da Influenza A/metabolismo , Domínios Proteicos , Esfingomielinas/metabolismo , Transferrinas/metabolismoRESUMO
With the aid of the single-virus tracking technique and the quantum dot-labeling strategy, the internalization of the pseudorabies virus (PrV) has been visualized in real time. The results demonstrate that macropinocytosis can be considered as a major pathway for PrV entering HeLa cells, which facilitates the development of therapeutics for virus-triggered diseases.
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Herpesvirus Suídeo 1/fisiologia , Pinocitose , Pontos Quânticos/química , Internalização do Vírus , Animais , Células HeLa/virologia , Humanos , Microscopia Confocal/métodos , Fosfatidilinositol 3-Quinase/metabolismo , Proteína Quinase C/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Nexinas de Classificação/metabolismo , Suínos , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Autophagy is closely related to virus-induced disease and a comprehensive understanding of the autophagy-associated infection process of virus will be significant for developing more effective antiviral strategies. However, many critical issues and the underlying mechanism of autophagy in virus entry still need further investigation. Here, this study unveils the involvement of autophagy in influenza A virus entry. The quantum-dot-based single-virus tracking technique assists in real-time, prolonged, and multicolor visualization of the transport process of individual viruses and provides unambiguous dissection of the autophagic trafficking of viruses. These results reveal that roughly one-fifth of viruses are ferried into cells for infection by autophagic machineries, while the remaining are not. A comprehensive overview of the endocytic- and autophagic-trafficking process indicates two distinct trafficking pathway of viruses, either dependent on Rab5-positive endosomes or autophagosomes, with striking similarities. Expressing dominant-negative mutant of Rab5 suggests that the autophagic trafficking of viruses is independent on Rab5. The present study provides dynamic, precise, and mechanistic insights into the involvement of autophagy in virus entry, which contributes to a better understanding of the relationship between autophagy and virus entry. The quantum-dot-based single-virus tracking is proven to hold promise for autophagy-related fundamental research.
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Autofagia/fisiologia , Vírus da Influenza A/metabolismo , Pontos Quânticos , Autofagossomos/metabolismo , Endossomos/metabolismo , Humanos , Transporte Proteico , Internalização do VírusRESUMO
When infecting host cells, influenza virus must move on microfilaments (MFs) at the cell periphery and then move along microtubules (MTs) through the cytosol to reach the perinuclear region for genome release. But how viruses switch from the actin roadway to the microtubule highway remains obscure. To settle this issue, we systematically dissected the role of related motor proteins in the transport of influenza virus between cytoskeletal filaments in situ and in real-time using quantum dot (QD)-based single-virus tracking (SVT) and multicolor imaging. We found that the switch between MF- and MT-based retrograde motor proteins, myosin VI (myoVI) and dynein, was responsible for the seamless transport of viruses from MFs to MTs during their infection. After virus entry by endocytosis, both the two types of motor proteins are attached to virus-carrying vesicles. MyoVI drives the viruses on MFs with dynein on the virus-carrying vesicle hitchhiking. After role exchanges at actin-microtubule intersections, dynein drives the virus along MTs toward the perinuclear region with myoVI remaining on the vesicle moving together. Such a "driver switchover" mechanism has answered the long-pending question of how viruses switch from MFs to MTs for their infection. It will also facilitate in-depth understanding of endocytosis.
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Citoesqueleto de Actina/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H9N2/fisiologia , Microtúbulos/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Citoesqueleto de Actina/patologia , Citoesqueleto de Actina/virologia , Animais , Cães , Dineínas/metabolismo , Endocitose , Células Madin Darby de Rim Canino , Microscopia Confocal , Microtúbulos/patologia , Microtúbulos/virologia , Cadeias Pesadas de Miosina/metabolismo , Imagem Óptica , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Internalização do VírusRESUMO
Entry is the first critical step for the infection of influenza A virus and of great significance for the research and development of antiflu drugs. Influenza A virus depends on exploitation of cellular endocytosis to enter its host cells, and its entry behaviors in distinct routes still need further investigation. With the aid of a single-virus tracking technique and quantum dots, we have realized real-time and multicolor visualization of the endocytic process of individual viruses and comprehensive dissection of two distinct dynamin-dependent endocytic pathways of influenza A virus, either dependent on clathrin or not. Based on the sequential progression of protein recruitment and viral motility, we have revealed the asynchronization in the recruitments of clathrin and dynamin during clathrin-dependent entry of the virus, with a large population of events for short-lived recruitments of these two proteins being abortive. In addition, the differentiated durations of dynamin recruitment and responses to inhibitors in these two routes have evidenced somewhat different roles of dynamin. Besides promoting membrane fission in both entry routes, dynamin also participates in the maturation of a clathrin-coated pit in the clathrin-dependent route. Collectively, the current study displays a dynamic and precise image of the entry process of influenza A virus and elucidates the mechanisms of distinct entry routes. This quantum dot-based single-virus tracking technique is proven to be well-suited for investigating the choreographed interactions between virus and cellular proteins.
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Rastreamento de Células/métodos , Endocitose/fisiologia , Internalização do Vírus/efeitos dos fármacos , Animais , Linhagem Celular , Clatrina/metabolismo , Cães , Dinaminas/metabolismo , Humanos , Vírus da Influenza A/patogenicidade , Células Madin Darby de Rim Canino , Pontos QuânticosRESUMO
Real-time, long-term, single-particle tracking (SPT) provides us an opportunity to explore the fate of individual viruses toward understanding the mechanisms underlying virus infection, which in turn could lead to the development of therapeutics against viral diseases. However, the research focusing on the virus assembly and egress by SPT remains a challenge because established labeling strategies could neither specifically label progeny viruses nor make them distinguishable from the parental viruses. Herein, we have established a temporally controllable capsid-specific HaloTag labeling strategy based on reverse genetic technology. VP26, the smallest pseudorabies virus (PrV) capsid protein, was fused with HaloTag protein and labeled with the HaloTag ligand during virus replication. The labeled replication-competent recombinant PrV harvested from medium can be applied directly in SPT experiments without further modification. Thus, virus infectivity, which is critical for the visualization and analysis of viral motion, is retained to the largest extent. Moreover, progeny viruses can be distinguished from parental viruses using diverse HaloTag ligands. Consequently, the entire course of virus infection and replication can be visualized continuously, including virus attachment and capsid entry, transportation of capsids to the nucleus along microtubules, docking of capsids on the nucleus, endonuclear assembly of progeny capsids, and the egress of progeny viruses. In combination with SPT, the established strategy represents a versatile means to reveal the mechanisms and dynamic global picture of the life cycle of a virus.
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Capsídeo/ultraestrutura , Herpesvirus Suídeo 1/ultraestrutura , Coloração e Rotulagem/métodos , Vírion/ultraestrutura , Replicação Viral/fisiologia , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Chlorocebus aethiops , Cricetinae , Corantes Fluorescentes/química , Expressão Gênica , Herpesvirus Suídeo 1/fisiologia , Camundongos , Microscopia de Fluorescência , Microtúbulos/ultraestrutura , Microtúbulos/virologia , Compostos Orgânicos/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Genética Reversa , Células Vero , Vírion/metabolismo , Montagem de Vírus/fisiologia , Liberação de Vírus/fisiologiaRESUMO
Real-time tracking of fluorophore-tagged viruses in living cells can help uncover virus infection mechanisms. Certainly, the indispensable prerequisite for virus-tracking is to label viruses with some bright and photostable beacons such as quantum dots (QDs) via an appropriate labeling strategy. Herein, we devise a convenient hydrazine-aldehyde based strategy to label viruses with QDs through the conjugation of 4-formylbenzoate (4FB) modified QDs to 6-hydrazinonicotinate acetone hydrazone (HyNic) modified viruses under mild conditions. On the basis of this strategy, viruses can be successfully labeled with QDs with high selectivity, stable conjugation, good reproducibility, high labeling efficiency of 92-93% and maximum retention of both fluorescence properties of QDs and infectivity of viruses, which is very meaningful to tracking and statistical analysis of virus infection processes. By further comparing with the most widely used labeling strategy based on the Biotin-SA system, this new strategy has advantages of both high labeling efficiency and good retention of virus infectivity, thus offering a promising alternative for virus-labeling. Moreover, due to the ubiquitous presence of exposed amino groups on the surface of various viruses, this selective, efficient, reproducible and biofriendly strategy should have good universality for labeling both enveloped and nonenveloped viruses.
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Aldeídos/química , Hidrazinas/química , Pontos Quânticos/química , Vírus/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Vírus da Influenza A Subtipo H9N2/química , Imagem Óptica , Pontos Quânticos/toxicidade , Coloração e Rotulagem , Células VeroRESUMO
Three-dimensional (3D) single-particle tracking (SPT) techniques have been widely reported. However, the 3D SPT technique remains poorly used for solving actual biological problems. In this work, a quantum dots (QDs)-based single-particle tracking technique is utilized to explore the Rab5- and Rab7-associated infection behaviors of influenza virus in three dimensions with a set of easily-attained equipment by the fast and accurate centroid method for 3D SPT. The experimental results indicate that Rab5 protein takes part in the virus infection process from the cell periphery to the perinuclear region, while Rab7 protein is mainly involved in the intermittent and confined movements of the virus in the perinuclear region. Evidently, the transition process of the virus-containing vesicles from early to late endosomes might occur during the intermittent movement in the perinuclear region. These findings reveal distinct dynamic behaviors of Rab5- and Rab7-positive endosomes in the course of the intracellular transport of viruses. This work is helpful in understanding the intracellular transport of cargoes.
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Influenza Humana/prevenção & controle , Proteínas rab de Ligação ao GTP/fisiologia , Proteínas rab5 de Ligação ao GTP/fisiologia , Animais , Cães , Humanos , Células Madin Darby de Rim Canino , proteínas de unión al GTP Rab7RESUMO
Understanding the microtubule-dependent behaviors of viruses in live cells is very meaningful for revealing the mechanisms of virus infection and endocytosis. Herein, we used a quantum dots-based single-particle tracking technique to dynamically and globally visualize the microtubule-dependent transport behaviors of influenza virus in live cells. We found that the intersection configuration of microtubules can interfere with the transport behaviors of the virus in live cells, which lead to the changing and long-time pausing of the transport behavior of viruses. Our results revealed that most of the viruses moved along straight microtubules rapidly and unidirectionally from the cell periphery to the microtubule organizing center (MTOC) near the bottom of the cell, and the viruses were confined in the grid of microtubules near the top of the cell and at the MTOC near the bottom of the cell. These results provided deep insights into the influence of entire microtubule geometry on the virus infection.
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Células/ultraestrutura , Células/virologia , Microtúbulos/ultraestrutura , Microtúbulos/virologia , Orthomyxoviridae/ultraestrutura , Animais , Cães , Endocitose , Humanos , Processamento de Imagem Assistida por Computador , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Microscopia de Fluorescência , Pontos Quânticos , Proteínas do Envelope Viral/químicaRESUMO
The study on circulating tumor cells (CTCs) has great significance for cancer prognosis, treatment monitoring, and metastasis diagnosis, in which isolation and enrichment of CTCs are key steps due to their extremely low concentration in peripheral blood. Herein, magnetic nanospheres (MNs) were fabricated by a convenient and highly controllable layer-by-layer assembly method. The MNs were nanosized with fast magnetic response, and nearly all of the MNs could be captured by 1 min attraction with a commercial magnetic scaffold. In addition, the MNs were very stable without aggregation or precipitation in whole blood and could be re-collected nearly at 100% in a monodisperse state. Modified with anti-epithelial-cell-adhesion-molecule (EpCAM) antibody, the obtained immunomagnetic nanospheres (IMNs) successfully captured extremely rare tumor cells in whole blood with an efficiency of more than 94% via only a 5 min incubation. Moreover, the isolated cells remained viable at 90.5 ± 1.2%, and they could be directly used for culture, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry (ICC) identification. ICC identification and enumeration of the tumor cells in the same blood samples showed high sensitivity and good reproducibility. Furthermore, the IMNs were successfully applied to the isolation and detection of CTCs in cancer patient peripheral blood samples, and even one CTC in the whole blood sample was able to be detected, which suggested they would be a promising tool for CTC enrichment and detection.
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Nanosferas , Neoplasias/sangue , Células Neoplásicas Circulantes , Antígenos de Neoplasias/imunologia , Sequência de Bases , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Primers do DNA , Molécula de Adesão da Célula Epitelial , Humanos , Separação Imunomagnética , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Exploring the virus infection mechanisms is significant for defending against virus infection and providing a basis for studying endocytosis mechanisms. Single-particle tracking technique is a powerful tool to monitor virus infection in real time for obtaining dynamic information. In this study, we reported a quantum-dot-based single-particle tracking technique to efficiently and globally research the virus infection behaviors in individual cells. It was observed that many influenza viruses were moving rapidly, converging to the microtubule organizing center (MTOC), interacting with acidic endosomes, and finally entering the target endosomes for genome release, which provides a vivid portrayal of the five-stage virus infection process. This report settles a long-pending question of how viruses move and interact with acidic endosomes before genome release in the perinuclear region and also finds that influenza virus infection is likely to be a "MTOC rescue" model for genome release. The systemic technique developed in this report is expected to be widely used for studying the mechanisms of virus infection and uncovering the secrets of endocytosis.
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Rastreamento de Células/métodos , Vírus da Influenza A Subtipo H9N2/ultraestrutura , Influenza Humana/patologia , Influenza Humana/virologia , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Pontos Quânticos , Células Cultivadas , Humanos , Vírion/ultraestruturaRESUMO
Wheat germ agglutinin (WGA) is a paradigm for understanding intracellular transport of lectins. As a protein exploiting the receptor-mediated endocytosis for internalization, WGA is also a valuable model system for exploring the endocytic and exocytic pathway. In this study, quantum dot-based single-particle tracking was performed to investigate the transport of WGA in live cells, revealing firstly that the endocytic and exocytic processes of WGA were both actin- and microtubule-dependent, each including five stages. The vesicle fusion event occurred near the cytomembrane, followed by two destinies with WGA: shedding to the extracellular or reversing to the cytoplasm. These findings suggest a distinct and dynamic scenario for the transport of lectins following a receptor-mediated endo/exocytic pathway in live cells. This is important for the application of lectins as drug carriers and antineoplastic drugs in medicine, and also offers insights into the pathway of endocytosis and exocytosis.